Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach

Abstract

Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

Figure 1

Properties of naked DNA loops attached to the NM. (A) Drawing illustrating the local topology along a typical supercoiled DNA loop that correlates with both distance relative to the NM and sensitivity to DNase I. (B) Phase contrast micrograph showing the NM of a hepatocyte nucleoid. (C) Fluorescence micrograph showing the DNA halo around the NM of a hepatocyte nucleoid caused by the unwinding of the supercoiled DNA loops by treatment of the nucleoid with the DNA-intercalating agent ethidium bromide (80 µg/ml). Scale bars 10 µm.

Figure 2

Kinetics of nucleoid DNA digestion. Nucleoids were digested with DNase-I (0.5 U/ml). Each time-point value is the average of separate experiments with samples obtained from separate animals (n = 9 for hepatocytes, n = 4 for naïve B-lymphocytes). Bars indicate the corresponding SD. The topological zones relative to the NM correspond to decreasing percentages of total DNA bound to the NM. (A) For hepatocytes: distal to NM (100–42% total DNA), proximal to NM (42–30% total DNA), very close to NM (30–19% total DNA) and embedded within NM (19–0% total DNA). The corresponding slopes are the following: 0–5 min = −9.80; 5–15 min = −1.70; 15–30 min = −0.47 and 30–60 min = −0.17. Hence, the slope of the digestion curve became close to zero by 60 min of DNase I treatment and remained like that even after 120 min of incubation with the enzyme (slope 60–120 min = −0.17). (B) For naïve B-lymphocytes: distal to NM (100–33% total DNA), proximal to NM (33–7% total DNA), very close to NM (7–4% total DNA) and embedded within NM (4–0% total DNA). The corresponding slopes are the following: 0–5 min = −11.46; 5–15 min = −2.84; 15–30 min = −0.61 and 30–60 min = −0.06 (slope 60–120 min = −0.03). Hence, the slope of the digestion curve also became close to zero by 60 min of DNase I treatment.

Figure 3

Procedure for mapping the position of DNA sequences relative to the NM. (A) Nucleoids prepared from freshly isolated rat primary cells were incubated with DNase I so as to progressively digest the loop DNA, obtaining a tri-phasic kinetics of digestion (B). Nucleoid samples with partially digested NM-bound DNA, as shown in the drawing and fluorescent micrographs, (C) were used for PCR amplification of target DNA sequences located along the genomic region containing members of the rat albumin gene family. The amplicons were run in agarose gels and scored as present (+) or absent (−) by an image-analysis software (A) as a function of nucleoid-sample digestion time (C).

Figure 4

The 162-kb genomic region containing members of the rat albumin gene family (chromosome 14): Alb (albumin), Afp (alpha-fetoprotein), Afm (afamin), Afp-L (pseudo-gene similar to Afp). The letters indicate the location of the target DNA sequences to be positionally mapped relative to the NM.

Figure 5

Positional mapping relative to the NM of specific target DNA sequences by PCR. Rat-hepatocyte nucleoids were treated with DNase-I (0.5 U/ml) for different times. The residual NM-bound DNA was directly used as template for PCR amplification of the target sequences (a–o). The specific amplicons were resolved in 2% agarose gels stained with ethidium bromide (0.5 µl/ml). C, control. Topological zones with respect to NM: D, distal; P, proximal; VC, very close; E, embedded within NM. (−), negative control (no template); (+), positive control (pure genomic DNA as template). The amplification patterns were consistently reproduced in separate experiments with samples from independent animals (n = 4).

Figure 6

Experimentally determined structural DNA loop organization of the 162-kb region containing members of the rat albumin gene family in hepatocytes (A) and naïve B-lymphocytes (B). The letters indicate the position of the target sequences within the DNA loops. The bold lines indicate the position of the whole named genes. The dashed lines indicate projected loop regions outside of the region studied. The left bar indicates the relative DNA loop size from tip to base; hence the whole DNA loop length is approximately the double of such value. The right bar indicates the topological zones relative to the NM according to the kinetics of nucleoid DNA digestion. The illustration is according to scale in kb along the x and y axis.